Evaluating the Effects of Fibrinogen αC Mutations on the Ability of Factor XIII to Crosslink the Reactive αC Glutamines (Q237, Q328, Q366)

Abstract

Fibrinogen (Fbg) levels and extent of fibrin polymerization have been associated with various pathological conditions such as cardiovascular disease, arteriosclerosis, and coagulation disorders. Activated factor XIII (FXIIIa) introduces γ-glutamyl-ε-lysinyl isopeptide bonds between reactive glutamines and lysines in the fibrin network to form a blood clot resistant to fibrinolysis. FXIIIa crosslinks the γ-chains and at multiple sites in the αC region of Fbg. Fbg αC contains a FXIII binding site involving αC (389–402) that is located near three glutamines whose reactivities rank Q237 >> Q366 ≈ Q328. Mass spectrometry and two-dimensional heteronuclear single-quantum correlation nuclear magnetic resonance assays were used to probe the anchoring role that αC E396 may play in controlling FXIII function and characterize the effects of Q237 on the reactivities of Q328 and Q366. Studies with αC (233–425) revealed that the E396A mutation does not prevent the transglutaminase function of FXIII A2 or A2B2. Other residues must play a compensatory role in targeting FXIII to αC. Unlike full Fbg, Fbg αC (233–425) did not promote thrombin cleavage of FXIII, an event contributing to activation. With the αC (233–425) E396A mutant, Q237 exhibited slower reactivities compared with αC wild-type (WT) consistent with difficulties in directing this N-terminal segment toward an anchored FXIII interacting at a weaker binding region. Q328 and Q366 became less reactive when Q237 was replaced with inactive N237. Q237 crosslinking is proposed to promote targeting of Q328 and Q366 to the FXIII active site. FXIII thus uses Fbg αC anchoring sites and distinct Q environments to regulate substrate specificity.

Transglutaminase Activities of Blood Coagulant Factor XIII Are Dependent on the Activation Pathways and on the Substrates

“…Electrostatic anchoring of Fbg αC E396 is proposed to play a contributing role in FXIII-A* reactivity. 26,28 A bigger hindrance to FXIII-A* occurred in this project when Fbg αC (233-425) residues beyond 388 were eliminated, thus removing the full FXIII-A*-binding region (αC 389-402). Physiologically, Fbg Keokuk (Q328stop) contains an even greater truncation leading to hypofibrinogenemia and postsurgical thrombosis and miscarriages.…”

“…Fbg αC (233–425) and variants were expressed in Escherichia coli and purified as described previously. 28 30…”

“…Fbg αC (233-425) and variants were expressed in Escherichia coli and purified as described previously. 28,30 Monodansylcadaverine Incorporation Assay A 2 μM rFXIII-A 2 was activated proteolytically with 50 nM recombinant human thrombin and 4 mM CaCl 2 and nonproteolytically with 100 mM CaCl 2 by incubating at 37°C for 30 minutes in Tris-buffered saline (TBS) buffer (50 mM tris acetate, 150 mM NaCl, pH 7.4). These conditions ensure full activation of FXIII in 30 minutes in both proteolytic and nonproteolytic assays.…”

“…24,25 FXIII-A* in the presence of Ca 2þ has been reported to bind to Fbg αC (389-402), with Fbg αC E396 serving as an important anchoring site. [26][27][28] Thus, Fbg αC (233-425) provides an effective substrate model for FXIII-A.…”

Functional and Structural Characterization of Nucleic Acid Ligands That Bind to Activated Coagulation Factor XIII

“…Specific details of FXIII interfaces are limited to only few handful studies and these studies are also limited in their exactness [ 36 , 40 ]. Most of these studies present only one dimensional aspect of these interactions, primarily because of the presence of multiple reactive glutamine donor residues on large substrates like fibrinogen [ 42 ]. Furthermore, these studies are heavily reliant on experimentally driven modeling approaches that leave room for other possibilities.…”